RESUMEN
The potassium (K+) ion channel KCNK13 is specifically expressed in human microglia with elevated expression observed in post-mortem human brain tissue from patients with Alzheimer's disease. Modulation of KCNK13 activity by a small-molecule inhibitor is proposed as a potential treatment for neurodegenerative diseases. Herein, we describe the evolution of a series of KCNK13 inhibitors derived from a high-throughput screening campaign, resulting in CVN293, a potent, selective, and brain permeable clinical candidate molecule. CVN293 demonstrated a concentration-dependent inhibition of the NLRP3-inflammasome mediated production of IL-1ß from LPS-primed murine microglia. Cross-species pharmacokinetic data of CVN293 are also disclosed. These findings support the advancement of CVN293 in clinical trials.
RESUMEN
Nicotinic acetylcholine receptor (nAChR) α6 subunit RNA expression is relatively restricted to midbrain regions and is located presynaptically on dopaminergic neurons projecting to the striatum. This subunit modulates dopamine neurotransmission and may have therapeutic potential in movement disorders. We aimed to develop potent and selective α6-containing nAChR antagonists to explore modulation of dopamine release and regulation of motor function in vivo. High-throughput screening (HTS) identified novel α6-containing nAChR antagonists and led to the development of CVN417. This molecule blocks α6-containing nAChR activity in recombinant cells and reduces firing frequency of noradrenergic neurons in the rodent locus coeruleus. CVN417 modulated phasic dopaminergic neurotransmission in an impulse-dependent manner. In a rodent model of resting tremor, CVN417 attenuated this behavioral phenotype. These data suggest that selective antagonism of α6-containing nAChR, with molecules such as CVN417, may have therapeutic utility in treating the movement dysfunctions observed in conditions such as Parkinson's disease.
Asunto(s)
Dopamina , Receptores Nicotínicos , Encéfalo , Membrana Celular , Cuerpo Estriado , Antagonistas Nicotínicos/farmacologíaRESUMEN
Neuroinflammation, specifically the NLRP3 inflammasome cascade, is a common underlying pathological feature of many neurodegenerative diseases. Evidence suggests that NLRP3 activation involves changes in intracellular K+. Nuclear Enriched Transcript Sort Sequencing (NETSseq), which allows for deep sequencing of purified cell types from human post-mortem brain tissue, demonstrated a highly specific expression of the tandem pore domain halothane-inhibited K+ channel 1 (THIK-1) in microglia compared to other glial and neuronal cell types in the human brain. NETSseq also showed a significant increase of THIK-1 in microglia isolated from cortical regions of brains with Alzheimer's disease (AD) relative to control donors. Herein, we report the discovery and pharmacological characterisation of C101248, the first selective small-molecule inhibitor of THIK-1. C101248 showed a concentration-dependent inhibition of both mouse and human THIK-1 (IC50: â¼50 nM) and was inactive against K2P family members TREK-1 and TWIK-2, and Kv2.1. Whole-cell patch-clamp recordings of microglia from mouse hippocampal slices showed that C101248 potently blocked both tonic and ATP-evoked THIK-1 K+ currents. Notably, C101248 had no effect on other constitutively active resting conductance in slices from THIK-1-depleted mice. In isolated microglia, C101248 prevented NLRP3-dependent release of IL-1ß, an effect not seen in THIK-1-depleted microglia. In conclusion, we demonstrated that inhibiting THIK-1 (a microglia specific gene that is upregulated in brains from donors with AD) using a novel selective modulator attenuates the NLRP3-dependent release of IL-1ß from microglia, which suggests that this channel may be a potential therapeutic target for the modulation of neuroinflammation in AD.
Asunto(s)
Enfermedad de Alzheimer , Inflamasomas , Canales de Potasio de Dominio Poro en Tándem , Animales , Humanos , Ratones , Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Inflamasomas/metabolismo , Microglía , Enfermedades Neuroinflamatorias , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidoresRESUMEN
Accumulation of tau is observed in dementia, with human tau displaying 6 isoforms grouped by whether they display either 3 or 4 C-terminal repeat domains (3R or 4R) and exhibit no (0N), one (1N) or two (2N) N terminal repeats. Overexpression of 4R0N-tau in rat hippocampal slices enhanced the L-type calcium (Ca2+) current-dependent components of the medium and slow afterhyperpolarizations (AHPs). Overexpression of both 4R0N-tau and 4R2N-tau augmented CaV1.2-mediated L-type currents when expressed in tsA-201 cells, an effect not observed with the third 4R isoform, 4R1N-tau. Current enhancement was only observed when the pore-forming subunit was co-expressed with CaVß3 and not CaVß2a subunits. Non-stationary noise analysis indicated that enhanced Ca2+ channel current arose from a larger number of functional channels. 4R0N-tau and CaVß3 were found to be physically associated by co-immunoprecipitation. In contrast, the 4R1N-tau isoform that did not augment expressed macroscopic L-type Ca2+ current exhibited greatly reduced binding to CaVß3. These data suggest that physical association between tau and the CaVß3 subunit stabilises functional L-type channels in the membrane, increasing channel number and Ca2+ influx. Enhancing the Ca2+-dependent component of AHPs would produce cognitive impairment that underlie those seen in the early phases of tauopathies.
Asunto(s)
Calcio , Tauopatías , Animales , Calcio/metabolismo , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/genética , Calcio de la Dieta/metabolismo , Hipocampo/metabolismo , Humanos , Neuronas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas , Tauopatías/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
Multielectrode arrays (MEAs) are grids of substrate-integrated microelectrodes that allow for electrophysiological interrogation of dissociated cell cultures or tissue slices. Here we discuss the use of nonimplantable electrodes for studies. The methods described attempt to provide a starting point for researchers new to the field who wish to begin to utilize this powerful, but daunting technology and quickly apply the basic principles to their own research interests.
Asunto(s)
Potenciales de Acción , Red Nerviosa/fisiología , Neuronas/fisiología , Análisis de Matrices Tisulares/instrumentación , Animales , Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Electrofisiología/instrumentación , Electrofisiología/métodos , Diseño de Equipo , Hipocampo/citología , Hipocampo/fisiología , Ratones , Microelectrodos , Red Nerviosa/citología , Neuronas/citología , Técnicas de Cultivo de Órganos/instrumentación , Técnicas de Cultivo de Órganos/métodos , Ratas , Análisis de Matrices Tisulares/métodosRESUMEN
Animals synchronize to the environmental day-night cycle by means of an internal circadian clock in the brain. In mammals, this timekeeping mechanism is housed in the suprachiasmatic nucleus (SCN) of the hypothalamus and is entrained by light input from the retina. One output of the SCN is a neural code for circadian time, which arises from the collective activity of neurons within the SCN circuit and comprises two fundamental components: 1) periodic alterations in the spontaneous excitability of individual neurons that result in higher firing rates during the day and lower firing rates at night, and 2) synchronization of these cellular oscillations throughout the SCN. In this review, we summarize current evidence for the identity of ion channels in SCN neurons and the mechanisms by which they set the rhythmic parameters of the time code. During the day, voltage-dependent and independent Na+ and Ca2+ currents, as well as several K+ currents, contribute to increased membrane excitability and therefore higher firing frequency. At night, an increase in different K+ currents, including Ca2+-activated BK currents, contribute to membrane hyperpolarization and decreased firing. Layered on top of these intrinsically regulated changes in membrane excitability, more than a dozen neuromodulators influence action potential activity and rhythmicity in SCN neurons, facilitating both synchronization and plasticity of the neural code.